A Dual-Color Electrophoretic Mobility Shift Assay to Examine RNA-Binding and RNA Chaperone Activity Simultaneously

RNA-binding proteins that rearrange RNA structure often undergo complicated reaction pathways including multivalent interactions with itself and the RNA substrates. For detailed mechanistic understanding of these reactions, it is essential to get insight into the individual complexes formed during the reaction that can guide answering questions regarding sequence or stoichiometry of the reaction. Meanwhile high-resolution structural approaches allow discrimination of multiple conformations and complexes in a heterogeneous sample; however, these approaches are cost- and time-intensive and require extensive technical experience. Traditional electrophoretic mobility shift assays are cheap and require no specialized equipment, and can give valuable insight into reaction pathways resolving both RNA-RNA and protein-RNA interactions.In this chapter, I present an adaptation of a gel electrophoretic mobility shift assay using individually fluorescently labelled RNAs in a discontinuous gel system that can resolve protein-RNA and RNA-RNA complexes from a single reaction in the same gel. The method is applied and discussed based on the example of the RNA-unwinding activity of the RNA helicase eIF4A1 including a small molecule eIF4A1-modulator silvestrol. The example demonstrates the capacity of the assay to provide detailed mechanistic insight into rearrangement of RNA structure and protein-RNA interactions.